Exploring Coumarin-Based Boron Emissive Complexes as Temperature Thermometers in Polymer-Supported Materials.

Three coumarin-based boron complexes (L1, L2 and L3) were designed and successfully incorporated into polymeric matrixes for evaluation as temperature probes. The photophysical properties of the complexes were carried out in different solvents and in the solid state. In solution, compound L1 exhibited the highest fluorescence quantum yield, 33%, with a positive solvatochromism also being observed on the absorption and emission when the polarity of the solvent increased. Additionally in the presence of anions, L1 showed a colour change from yellow to pink, followed by a quenching in the emission intensity, which is due to deprotonation with the formation of a quinone base. Absorption and fluorescence spectra of L1 were calculated at different temperatures by the DFT/B3LYP method. The decrease in fluorescence of compound L1 with an increase in temperature seems to be due to the presence of pronounced torsional vibrations of the donor and acceptor fragments relative to the single bond with C(carbonyl)-C (styrene fragment). L1, L2 and L3, through their incorporation into the polymeric matrixes, became highly emissive by aggregation. These dye@doped polymers were evaluated as temperature sensors, showing an excellent fluorescent response and reversibility after 15 cycles of heating and cooling.

Development of New Targeted Nanotherapy Combined with Magneto-Fluorescent Nanoparticles against Colorectal Cancer.

The need for non-invasive therapies capable of conserving drug efficiency and stability while having specific targetability against colorectal cancer (CRC), has made nanoparticles preferable vehicles and principal building blocks for the development of complex and multi-action anti-tumoral approaches. For that purpose, we herein report the production of a combinatory anti-tumoral nanotherapy using the production of a new targeting towards CRC lines. To do so, Magneto-fluorescent NANO3 nanoparticles were used as nanocarriers for a combination of the drugs doxorubicin (DOX) and ofloxacin (OFLO). NANO3 nanoparticles' surface was modified with two different targeting agents, a newly synthesized (anti-CA IX acetazolamide derivative (AZM-SH)) and a commercially available (anti-epidermal growth factor receptor (EGFR), Cetuximab). The cytotoxicity revealed that only DOX-containing nanosystems showed significant and even competitive cytotoxicity when compared to that of free DOX. Interestingly, surface modification with AZM-SH promoted an increased cellular uptake in the HCT116 cell line, surpassing even those functionalized with Cetuximab. The results show that the new target has high potential to be used as a nanotherapy agent for CRC cells, surpassing commercial targets. As a proof-of-concept, an oral administration form of NANO3 systems was successfully combined with Eudragit® enteric coating and studied under extreme conditions.

Magneto-Fluorescent Mesoporous Nanocarriers for the Dual-Delivery of Ofloxacin and Doxorubicin to Tackle Opportunistic Bacterial Infections in Colorectal Cancer.

Cancer-related opportunistic bacterial infections are one major barrier for successful clinical therapies, often correlated to the production of genotoxic factors and higher cancer incidence. Although dual anticancer and antimicrobial therapies are a growing therapeutic fashion, they still fall short when it comes to specific delivery and local action in in vivo systems. Nanoparticles are seen as potential therapeutic vectors, be it by means of their intrinsic antibacterial properties and effective delivery capacity, or by means of their repeatedly reported modulation and maneuverability. Herein we report on the production of a biocompatible, antimicrobial magneto-fluorescent nanosystem (NANO3) for the delivery of a dual doxorubicin-ofloxacin formulation against cancer-related bacterial infections. The drug delivery capacity, rendered by its mesoporous silica matrix, is confirmed by the high loading capacity and stimuli-driven release of both drugs, with preference for tumor-like acidic media. The pH-dependent emission of its surface fluorescent SiQDs, provides an insight into NANO3 surface behavior and pore availability, with the SiQDs working as pore gates. Hyperthermia induces heat generation to febrile temperatures, doubling drug release. NANO3-loaded systems demonstrate significant antimicrobial activity, specifically after the application of hyperthermia conditions. NANO3 structure and antimicrobial properties confirm their potential use in a future dual anticancer and antimicrobial therapeutical vector, due to their drug loading capacity and their surface availability for further modification with bioactive, targeting species.

Ultrasonic-Based Filter Aided Sample Preparation as the General Method to Sample Preparation in Proteomics.

We propose a new high-throughput ultrafast method for large-scale proteomics approaches by speeding up the classic filter aided sample preparation protocol, FASP, from overnight to 2.5 h. Thirty-six samples can be treated in 2.5 h, and the method is scalable to 96-well plate-based pipelines. After a modification of the FASP-tube, the steps of protein reduction, protein alkylation, and protein digestion of complex proteomes are done in just 5.25 min, each one under the effects of an ultrasonic field (7 cycles: 30 s on and 15 s off). The new method was compared to the standard overnight digestion FASP protocol, and no statistical differences were found for more than 92.4%, 92%, and 93.3% of the proteins identified by studying the proteome of E. coli, mouse brain, and mouse liver tissue samples, respectively. Furthermore, the successful relative label-free quantification of four spiked proteins in E. coli samples, BSA, ß-lactoglobulin, α-casein, and α-lactalbumin, was achieved, using either the ultrasonic-based FASP protocol or the classic overnight one. The new US-FASP method matches the analytical minimalism rules as time, cost, sample requirement, reagent consumption, energy requirements, and production of waste products are reduced to a minimum while maintaining high sample throughput in a robust manner as all of the advantages of the filter aided sample preparation protocol are maintained.

Lights and colours: Science, Techniques and Surveillance for the Future - 4th IC3EM 2020, Caparica, Portugal.

•Special Issue on 4th IC3EM 2020.•Science, Techniques, Surveillance.•The importance of Science.•Fluorescent dyes.

Label-free quantitative mass spectrometry from formalin-fixed paraffin-embedded samples of nasopharyngeal carcinoma: Preliminary results from a non-endemic European cohort of patients.

AIM: Report our results of biomarker discovery in formalin-fixed paraffin-embedded (FFPE) nasopharyngeal carcinoma (NPC) via proteomic analysis. BACKGROUND: Nasopharyngeal carcinoma (NPC) is a rare cancer in Western countries. Proteomic analysis have already been reported as a useful tool to provide biomarkers. Formalin-fixed paraffin-embedded (FFPE) samples, despite largely underused, can provide invaluable information for biomarker research via proteomic analysis. METHODS: FFPE samples of NPC were submitted to protein extraction followed by FASP-digestion and label-free quantitative mass spectrometry (MS). Patients' received concurrent chemoradiation with or without adjuvant chemotherapy as per Intergroup 0099 trial. IMRT was delivered following the RTOG0615 specifications. Toxicity was scored using the CTCAE 4.03 tables. Survival was estimated using Kaplan-Meier curves. Log-rank was used to detect differences. KEGG ontology graphics were generated. RESULTS: 28 FFPE samples from NPC patients were used. Patients were: 79% male, 97% Caucasians, 86% WHO type 3, 40% T1, 10% T2, 25% T3, and 25% T4. With a median follow up of 37 months, local control was 83 (T1, 100% T2, T3 and T4), overall survival was 84%, and six patients developed distant metastases. All five patients that died were due to metastatic disease. Tumor samples contained a median of 75% of tumor material. We found Epstein-Barr (EBV) and Herpes simplex (HSV) viruses' related proteins significantly present in early-stage primary NPC (T1 and T2, p < 0.01). A pool of 10 proteins was statistically up-regulated in the metastatic group of patients (p < 0.01). Median survival from this M1 group was <1 year (p < 0.001). CONCLUSIONS: FFPE samples yielded adequate material for MS analysis. We found EBV and HSV related proteins on early-stage NPC, and proteomic profiling associated with distant metastases, potential candidates of disease biomarkers. Validation is needed.

Tailoring Mesoporous Silica-Coated Silver Nanoparticles and Polyurethane-Doped Films for Enhanced Antimicrobial Applications.

The global increase in multidrug-resistant bacteria poses a challenge to public health and requires the development of new antibacterial materials. In this study, we examined the bactericidal properties of mesoporous silica-coated silver nanoparticles, varying the core sizes (ca. 28 nm and 51 nm). We also investigated gold nanoparticles (ca. 26 nm) coated with mesoporous silica as possible inert metal cores. To investigate the modification of antimicrobial activity after the surface charge change, we used silver nanoparticles with a silver core of 28 nm coated with a mesoporous shell (ca. 16 nm) and functionalized with a terminal amine group. Furthermore, we developed a facile method to create mesoporous silica-coated silver nanoparticles (Ag@mSiO2) doped films using polyurethane (IROGRAN®) as a polymer matrix via solution casting. The antibacterial effects of silver nanoparticles with different core sizes were analyzed against Gram-negative and Gram-positive bacteria relevant to the healthcare and food industry. The results demonstrated that gold nanoparticles were inert, while silver nanoparticles exhibited antibacterial effects against Gram-negative (Escherichia coli and Salmonella enterica subsp. enterica serovar Choleraesuis) and Gram-positive (Bacillus cereus) strains. In particular, the larger Ag@mSiO2 nanoparticles showed a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 18 µg/mL in the Salmonella strain. Furthermore, upon terminal amine functionalization, reversing the surface charge to positive values, there was a significant increase in the antibacterial activity of the NPs compared to their negative counterparts. Finally, the antimicrobial properties of the nanoparticle-doped polyurethane films revealed a substantial improvement in antibacterial efficacy. This study provides valuable information on the potential of mesoporous silica-coated silver nanoparticles and their applications in fighting multidrug-resistant bacteria, especially in the healthcare and food industries.

Low Cubilin/Myeloperoxidase ratio as a promising biomarker for prognosis of high-grade T1 bladder cancer.

PURPOSE: T1 bladder cancer is known for its high progression and recurrence rates. Identifying aggressive tumours at the non-muscle-invasive stage is crucial to allow early interventions and subsequently increase patient survival. This study aimed to investigate the potential of the cubilin/myeloperoxidase (CUBN/MPO) ratio as a high-grade T1 bladder cancer biomarker. METHODS: Urine samples were collected from 30 patients who underwent transurethral resection of the tumour with high-grade T1 bladder cancer (June 2015 to December 2019) before surgery. The urinary proteome was analysed using high-resolution mass spectrometry and the CUBN/MPO ratio was calculated. The primary outcome was the recurrence during the follow-up (around 31.5 months after resection). Univariate Cox regression and Kaplan-Meier curves were used for data analysis. RESULTS: Patients with a low CUBN/MPO ratio exhibited upregulated MPO and/or downregulated CUBN. This group of patients had a higher incidence of disease recurrence and progression. Low CUBN/MPO ratio was significantly associated with a higher likelihood of recurrence, progression, and death. It is worth noting that this study was exploratory and conducted on a small sample size, so further research is needed to validate these findings in larger cohorts. CONCLUSION: This study highlights the potential of the CUBN/MPO ratio as a prognostic biomarker for high-grade T1 bladder cancer.

Clonal diversity of ESBL-producing Escherichia coli in pigs at slaughter level in Portugal.

We aimed to determine the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in fecal samples of healthy pigs, and to evaluate their clonality and associated resistance. Forty-nine percent of pigs sampled (n=35/71) in a slaughterhouse in Portugal revealed ESBL-producing E. coli isolates. Most isolates produced CTX-M-1 enzyme (71.4%; n=25/35), followed by CTX-M-9 (11.4%; n=4/35), CTX-M-14 (5.7%; n=2/35), SHV-12 (5.7%; n=2/35), and CTX-M-32 (5.7%; n=2/35). Ninety-four percent of the isolates presented a phenotype of multi-resistance. Most isolates belonged to phylogroups B1 (42.8%; n=15/35) and A (40%; n=14/35). Multilocus sequence typing (MLST) analysis revealed nine sequence types (STs) under six clonal complexes (CCs) and nine singletons, including overrepresentation of CC10 and three new STs (ST2524, ST2525, ST2528). We observed the frequent presence of CTX-M-producing E. coli in pigs at slaughter level, most of them belonging to CC10, commonly recovered from clinical samples.

High prevalence of antimicrobial-resistant Escherichia coli from animals at slaughter: a food safety risk.

BACKGROUND: There has been concern about the increase of antimicrobial resistant bacteria and protection of animal and public health, along with food safety. In the present study, we evaluate the incidence of antimicrobial resistance among 192 strains of Escherichia coli isolated from faecal samples of healthy food-producing animals at slaughter in Portugal. RESULTS: Ninety-seven % of the pig isolates, 74% from sheep and 55% from cattle were resistant to one or more antimicrobial agents, with the resistances to ampicillin, streptomycin, tetracycline and trimethoprim-sulfamethoxazole the most common phenotype detected. Genes encoding resistance to antimicrobial agents were detected in most of the resistant isolates. Ninety-three % of the resistant isolates were included in the A or B1 phylogenetic groups, and the virulence gene fimA (alone or in association with papC or aer genes) was detected in 137 of the resistant isolates. Five isolates from pigs belonging to phylogroup B2 and D were resistant to five different antimicrobial agents. CONCLUSION: Our data shows a high percentage of antibiotic resistance in E. coli isolates from food animals, and raises important questions in the potential impact of antibiotic use in animals and the possible transmission of resistant bacteria to humans through the food chain.

Copper(i) as a reducing agent for the synthesis of bimetallic PtCu catalytic nanoparticles.

This work investigates the potential utilization of Cu(i) as a reducing agent for the transformation of the platinum salt K2PtCl4, resulting in the production of stable nanoparticles. The synthesized nanoparticles exhibit a bimetallic composition, incorporating copper within their final structure. This approach offers a convenient and accessible methodology for the production of bimetallic nanostructures. The catalytic properties of these novel nanomaterials have been explored in various applications, including their use as artificial metalloenzymes and in the degradation of dyes. The findings underscore the significant potential of Cu(i)-mediated reduction in the development of functional nanomaterials with diverse catalytic applications.

Synthesis and Structural Characterization of Branched Bimetallic AuPd Nanoparticles with a Highly Tunable Optical Response.

Bimetallic nanostructures composed of gold (Au) and palladium (Pd) have garnered increased interest for their applications in heterogeneous catalysis. This study reports a simple strategy for manufacturing Au@Pd bimetallic branched nanoparticles (NPs), which offer a tunable optical response, using polyallylamine-stabilized branched AuNPs as template cores for Pd overgrowth. The palladium content can be altered by manipulating the concentration of PdCl42- and ascorbic acid (AA) that are injected, which permit an overgrowth of the Pd shell up to ca. 2 nm thick. The homogeneous distribution of Pd at the surfaces of Au NPs can be carried out regardless of their size or branching degree, which allows for an adjustment of the plasmon response in the near-infrared (NIR) spectral range. As a proof of concept, the nanoenzymatic activity of pure gold and gold-palladium NPs was compared, exploring their peroxidase-like activity in the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB). The bimetallic AuPd NPs demonstrate an increase in the catalytic properties attributed to the presence of palladium at the surface of gold.

Sequential depletion of human serum for the search of osteoarthritis biomarkers.

BACKGROUND: The field of biomarker discovery, development and application has been the subject of intense interest and activity, especially with the recent emergence of new technologies, such as proteomics-based approaches. In proteomics, search for biomarkers in biological fluids such as human serum is a challenging issue, mainly due to the high dynamic range of proteins present in these types of samples. Methods for reducing the content of most highly abundant proteins have been developed, including immunodepletion or protein equalization. In this work, we report for the first time the combination of a chemical sequential depletion method based in two protein precipitations with acetonitrile and DTT, with a subsequent two-dimensional difference in-gel electrophoresis (2D-DIGE) analysis for the search of osteoarthritis (OA) biomarkers in human serum. The depletion method proposed is non-expensive, of easy implementation and allows fast sample throughput. RESULTS: Following this workflow, we have compared sample pools of human serum obtained from 20 OA patients and 20 healthy controls. The DIGE study led to the identification of 16 protein forms (corresponding to 14 different proteins) that were significantly (p < 0.05) altered in OA when compared to controls (8 increased and 7 decreased). Immunoblot analyses confirmed for the first time the increase of an isoform of Haptoglobin beta chain (HPT) in sera from OA patients. CONCLUSIONS: Altogether, these data demonstrate the utility of the proposed chemical sequential depletion for the analysis of sera in protein biomarker discovery approaches, exhibit the usefulness of quantitative 2D gel-based strategies for the characterization of disease-specific patterns of protein modifications, and also provide a list of OA biomarker candidates for validation.

Validation of a Standard Luminescence Method for the Fast Determination of the Antimicrobial Activity of Nanoparticles in Escherichia coli.

The use of nanoparticles in multiple industries has raised concerned voices about the assessment of their toxicity/antimicrobial activity and the development of standardized handling protocols. Issues emerge during the antimicrobial assaying of multiple cargo, colorimetric, colloidal nanoformulations, as standard protocols often rely on visual evaluations, or optical density (OD) measurements, leading to high variance inhibitory concentrations (MIC). Thus, a fast, luminescence-based assay for the effective assessment of the antimicrobial activity of nanoparticles is herein reported, using the bioluminescence of an in-house E. coli ATCC® 8739TM construct with the pMV306G13 + Lux plasmid (E. coli Lux). The new strain's sensitivity to ofloxacin as a standard antibiotic was confirmed, and the methodology robustness verified against multiple nanoparticles and colorimetric drugs. The reduction of incubation from 24 to only 8 h, and the sole use of luminescence (LUX490) to accurately determine and distinguish MIC50 and MIC90, are two main advantages of the method. By discarding OD measurements, one can avoid turbidity and color interferences when calculating bacterial growth. This approach is an important tool that contributes to the standardization of methods, reducing samples' background interference and focusing on luminescence as a direct probe for bacterial metabolic activity, growth and, most importantly, the correct assessment of nanomaterials' antimicrobial activity.

Post-Global Pandemic Challenges and improvements in advanced detection and removal processes of toxic pollutants: Editorial.

Every two years, the Pollutant Toxic Ions and Molecules Conference, PTIM, meets the environmentalist, biologist, chemists and health researchers in Costa de Caparica, Portugal, to showcase the latest technologies, methodologies and research advances in pollution detection, contamination control, remediation, and related health issues, as well as policy implications.

Functional Cyanine-Based PVA:PVP Polymers as Antimicrobial Tools toward Food and Health-Care Bacterial Infections.

The rising of multidrug-resistant bacteria and their associated proliferation as harmful microorganisms boosts the creation of new antibacterial surfaces and biomaterials with applications ranging from health to food packing. Herein, low-cost antibacterial PVA:PVP copolymers containing cyanine derivatives (1, 2, and 3) and their respective Cu2+ complexes are successfully obtained and tested against Gram-negative and Gram-positive bacteria. The possible application in food packing is addressed by covering the surface of typical paper mockups with the doped polymers. All dye-doped polymers present a broad-spectrum antibacterial effect against Gram-positive bacteria, especially for Bacillus cereus (B. cereus), Staphylococcus aureus (S. aureus), and methicillin-resistant S. aureus (MRSA) strains, with PVA:PVP@3 and PVA:PVP@3-Cu being the most effective. Moreover, polymers containing cyanine derivatives present interesting inhibition effects against Pseudomonas aeruginosa (P. aeruginosa), where the production of its characteristic blue/green virulent pigment is not observed. Of the coated paper mockups, PVA:PVP:paper@2 and PVA:PVP:paper@2-Cu are most effective against B. cereus and S. aureus, while PVA:PVP:paper@3 and PVA:PVP:paper@3-Cu are most effective against the MRSA strain. In these formulations, direct contact inhibition mechanisms appear to be more significant than diffusional mechanisms, due to cyanine release hindrance, making them very interesting and versatile platforms for medical and food applications.

A comparison of depletion versus equalization for reducing high-abundance proteins in human serum.

In this work three methods to diminish the content of most highly abundant proteins in human serum have been studied and compared. Protein depletion with ACN or DTT and protein equalization with the ProteoMiner(™) (PM) have been assessed by 1-D gel electrophoresis and MS. After treatment 5, 18 and 9 major proteins within the 20 most abundant proteins in serum were identified for the ACN, DTT and PM methods, respectively. The ACN method was efficient for depleting high molecular weight proteins, over 75 KDa, resulting in 10±4% (n=3) of the total protein content remaining in the depleted serum. In addition, 75% of the proteins belonging to the group of the 20 most abundant proteins were not detected, making this depletion strategy a cheap alternative to expensive commercial tools regularly used for removing high abundance proteins from serum. The ACN extract was found rich in apolipoproteins. The dithithreitol method promotes the precipitation of proteins rich in disulfide bonds, mainly albumin, with 73±7% (n=3) of the total protein content remaining in the depleted serum, which was found rich in immunoglobulins. The PM method compresses the dynamic range of the serum proteins, rendering an extract containing 16±2% (n=3) of the total initial protein content. The extract was found to be rich in both apolipoproteins and immunoglobulins. As a general rule the DTT and PM methods provide a compression of the dynamic range of serum protein concentrations while the ACN method allows an effective depletion of the protein fraction above 72 KDa.

Impacts of environmental issues on health and well-being: a global pollution challenge.

Every 2 years, the environmental, chemical, and health research communities meet in Costa de Caparica, Portugal to showcase the latest technologies, methodologies and research advances in pollution detection, contamination control, remediation, and related health issues. Since its inception in 2015, the International Caparica Conference on Pollution Metal Ions and Molecules (PTIM) has become a biennial global forum to hear from those who protect the land, the water, and the air at all environmental scales. During past PTIM editions, we have learned about numerous efforts to develop new recovery and clean-up processes to restore the natural equilibria of our planet. Soil, land, water, and air are the key focus of efforts that will require deeper understanding and better control.

Synthesis of Mesoporous Silica Coated Gold Nanorods Loaded with Methylene Blue and Its Potentials in Antibacterial Applications.

In this work, the successful preparation and characterization of gold nanorods (AuNRs) coated with a mesoporous silica shell (AuNRs@Simes) was achieved. Conjugation with methylene blue (MB) as a model drug using ultrasound-stimulated loading has been explored for further application in light-mediated antibacterial studies. Lyophilization of this conjugated nanosystem was analyzed using trehalose (TRH) as a cryogenic protector. The obtained stable dry formulation shows potent antimicrobial activity against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria after a simple post-treatment irradiation method with a red laser during a short time period.

A novel (18)O inverse labeling-based workflow for accurate bottom-up mass spectrometry quantification of proteins separated by gel electrophoresis.

In the present work we report on a novel and fast protocol for accurate bottom-up protein quantification that overcomes the drawbacks of in-gel digestion and MALDI analysis, while maintaining their benefits. It relies on the following steps: (i) gel electrophoresis separation of proteins, (ii) fast in-gel protein digestion with trypsin, (iii) (18)O-labeling through the decoupled method, (iv) quantification through selected peptides previously chosen using the (18)O inverse labeling approach and that, finally, (v) it takes advantage of software specifically developed to select the peptides that will drive the quantification of the protein in an automated mode. We have accurately quantified the following six proteins: glycogen phosphorylase, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor, and α-lactalbumin. As a case study we have quantified the protein vitellogenin in plasma of Cyprinus carpio exposed to high levels of estrogens. The proposed new protocol was validated against the traditional ELISA method; both were found to provide comparable results (non-parametric Mann-Whitney U-test).